SIST EN ISO 9167-1:1998
(Main)Rapeseed - Determination of glucosinolates content - Part 1: Method using high-performance liquid chromatography (ISO 9167-1:1992)
Rapeseed - Determination of glucosinolates content - Part 1: Method using high-performance liquid chromatography (ISO 9167-1:1992)
This part of ISO 9167 specifies a method for the determination of the content of the different glucosinolates in rapeseeds (colza) using high- Performance liquid chromatography. NOTES 1 This method does not determine gfucosinolates which are substituted on the glucose molecule, but these compounds are of little importante in commercial rapeseed. 2 A rapid method for the determination of giucosinolates content using X-ray fluorescence spectrometry is the subject of ISO 9167-2.
Rapssamen - Bestimmung des Glucosinolatgehaltes - Teil 1: HPLC-Verfahren (ISO 9167-1:1992)
In diesem Teil von ISO 9167 wird ein Verfahren zur Bestimmung der verschiedenen Glucosinolatgehalte in Rapssamen (Kolza) mittels Hochleistungsflüssigkeitschromatographie festgelegt. Anmerkung 1: Glucosinolate, die am Glucose-Molekül substituiert sind, werden mit diesem Verfahren nicht erfaßt, sind für handelsübliche Rapssamen jedoch auch von untergeordneter Bedeutung. Anmerkung 2: Ein Schnellverfahren zur Bestimmung der Anteile der Glucosinolate unter Anwendung der Röntgenfluoreszensspektroskopie ist Thema von ISO 9267-2.
Graines de colza - Dosage des glucosinolates - Partie 1: Méthode par chromatographie liquide a haute performance (ISO 9167-1:1992)
La présente partie de l'ISO 9167 prescrit une méthode de dosage par chromatographie liquide à haute performance (CLHP) des différents glucosinolates dans les graines de colza.
NOTES 1 Cette méthode ne dose pas les glucosinolates ayant des substituants sur la partie glucose, mais ces composés sont peu importants dans les graines de colza commercialisées. 2 Une méthode rapide de dosage global des glucosinolates des graines de colza, par spectrométrie de fluorescence aux rayons X (XRF) fait l'objet de l'ISO 9167-2.
Seme oljne repice - Določevanje glukozinolatov - 1. del: Metoda s tekočinsko kromatografijo visoke ločljivosti (ISO 9167-1:1992)
Ta del ISO 9167 določa metodo za določevanje različnih glukozinolatov v oljni repici (repno seme) s tekočinsko kromatografijo visoke ločljivosti. OPOMBA 1: Ta metoda ne določa glukozinolatov, ki so v molekuli glukoze nadomeščeni, vendar te spojine v okviru komercialne oljne repice nimajo velikega pomena. OPOMBA 2: ISO 9167-2 opisuje hitro metodo določevanja glukozinolatov z rentgensko fluorescenčno spektrometrijo.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 9167-1:1998
01-november-1998
6HPHROMQHUHSLFH'RORþHYDQMHJOXNR]LQRODWRYGHO 0HWRGDVWHNRþLQVNR
NURPDWRJUDILMRYLVRNHORþOMLYRVWL ,62
Rapeseed - Determination of glucosinolates content - Part 1: Method using high-
performance liquid chromatography (ISO 9167-1:1992)
Rapssamen - Bestimmung des Glucosinolatgehaltes - Teil 1: HPLC-Verfahren (ISO 9167
-1:1992)
Graines de colza - Dosage des glucosinolates - Partie 1: Méthode par chromatographie
liquide a haute performance (ISO 9167-1:1992)
Ta slovenski standard je istoveten z: EN ISO 9167-1:1995
ICS:
67.200.20 Oljnice Oilseeds
SIST EN ISO 9167-1:1998 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 9167-1:1998
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SIST EN ISO 9167-1:1998
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SIST EN ISO 9167-1:1998
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SIST EN ISO 9167-1:1998
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SIST EN ISO 9167-1:1998
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SIST EN ISO 9167-1:1998
INTERNATIONAL
91674
STANDARD
First edition
1992-07-0 1
- ----
Rapeseed - Determination of glucosinolates
content -
Part 1:
Method using high-performance liquid
chromatography
Graines de colza - Dosage des qlucosinolates -
L
Partie 1: Methode par chromatographie liquide 3 haute perforrnance
~---- ---
--
Reference number
ISO 9167-1:1992(E)
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SIST EN ISO 9167-1:1998
ISO 91674:1992(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide
federation of national Standards bodies (ISO member bodies). The work
of preparing International Standards is normally carried out through ISO
technical committees. Esch member body interested in a subject for
which a technical committee has been established has the right to be
represented on that committee. International organizations, govern-
mental and non-governmental, in liaison with ISO, also take part in the
work. ISO collaborates closely with the International Electrotechnical
Commission (IEC) on all matters of electrotechnical standardization.
Draft International Standards adopted by the technical committees are
circulated to the member bodies for voting. Publication as an Inter-
national Standard requires approval by at least 75 % of the member
bodies casting a vote.
International Standard ISO 9167-1 was prepared by Technical Committee
ISO/TC 34, Agricultural food products, Sub-Committee SC 2, Oleaginous
seeds and fruits.
ISO 9167 consists of the following Parts, under the general title
Rapeseed - Determination of glucosinolates content:
-
Part 1: Method using high-performance liquid chromatography
-
Part 2: Method using X-ray fluorescence spectrometry
Annex A of this part of ISO 9167 is for information only.
0 ISO 1992
All rights reserved. No part of this publication may be reproduced or utilized in any form
or by any means, electronie or mechanical, including photocopylng and microfilm, without
Permission in writing from the publisher.
Intern ational Organizati on for Standardiz ation
211 Gen&e 20 * Switz erland
Case Postale 56 l CH-l
Printed in Switzerland
ii
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SIST EN ISO 9167-1:1998
ISO 91674:1992(E)
INTERNATIONAL STANDARD
- Determination of glucosinolates content -
Rapeseed
Part 1:
Method using high-performance liquid chromatography
1 Scope 3 Principle
Extraction of glucosinolates by methanol, then puri-
This part of ISO 9167 specifies a method for the
fication and enzymatic desulfatation on ion-
determination of the content of the different gluco-
exchange resins. Determination using
sinolates in rapeseeds (colza) using high-
reversed-Phase
high-performance liquid chro-
Performance liquid chromatography.
matography (HPLC) with elution gradient and ultra-
NOTES
violet detection.
1 This method does not determine gfucosinolates which
are substituted on the glucose molecule, but these com-
4 Reagents
pounds are of little importante in commercial rapeseed.
2 A rapid method for the determination of giucosinolates
Use only reagents of recognized analytical grade,
content using X-ray fluorescence spectrometry is the
unless otherwise specified, and water complying
subject of ISO 9167-2.
with grade 2 of ISO 3696.
4.1 Methanol, HPLC grade, 70 % (I/!I/) Solution.
4.2 Sodium acetate, 0,02 mol/1 at pH 4,0.
2 Normative references
4.3 Sodium acetate, 0,2 mol/1 solution.
The following Standards contain provisions which,
through reference in this text, constitute provisions
of this part of ISO 9167. At the time of publication,
4.4 Imidazofe formate, 6 mol/1 Solution.
the editions indicated were valid. All Standards are
subject to revision, and Parties to agreements based
Dissolve 204 g of imidazole in 113 ml of formic acid
on this part of ISO 9167 are encouraged to investi-
in a 500 ml one-mark volumetric flask. Make up to
gate the possibility of applying the most recent edi-
the mark with water.
tions of the Standards indicated below. Members of
IEC and ISO maintain registers of currently valid In-
ternational Standards.
4.5 lnternal Standard, use either sinigrin mono-
hydrate (potassium allylglucosinolate monohydrate,
ISO 664:1990, Oilseeds -- Reduction of laboratory
M, = 415,49) (see 4.5.1) or, for rapeseed (cultivated
Sample to fest Sample.
or self-propagated) in which sinigrin is present na-
turally, glucotropaeolin (benzylglucosinolate, pot-
Determination of moisture
ISO 665:1977, Oilseeds - assium salt, M, = 447,52) (see 4.5.2).
and volati/e matter content.
For rapeseed with a low glucosinolate content
(< 20 pm/g), reduce the internal Standard concen-
ISO 3696:1987, Water for analytical laboratory use -
tration (1 mmol/l to 3 mmol/l) in 4.5.1 and 4.5.2.1 .
Specification and test methods.
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SIST EN ISO 9167-1:1998
ISO 91674:1992(E)
4.5.1 Sinigrin monohydrate 4.5.2 Glucotropaeolin
NOTE 3 Glucotropaeolin is sometimes difficult to sep-
arate from other natura/ minor glucosinolates.
4.5.1.1 Sinigrin monohydrate, 5 mol/1 Solution.
4.5.2.1 Glucotropaeolin, 5 mmol/l Solution.
Dissolve 207,7 mg of potassium allylglucosinolate
monohydrate in water in a 100 ml one-mark
Dissolve 233,8 mg of glucotropaeolin in water in a
volumetric flask. Make \JP, to the mark with water.
100 mf volumetric flask. Make up to the mark with
water.
The Solution thus prepared may be stored in a
refrigerator at approximately 4 “C for up to a week
4.5.2.2 Glucotropaeolin, 20 mmol/t Solution.
or in a freezer at -
18 “C for a langer period.
Dissolve 895,0 mg of glucotropaeolin in water in a
100 ml volumetric flask. Make up to the mark with
water.
451.2 Sinigrin monohydrate, 20 mmol/l Solution.
4.5.2.3 Purity check
Dissolve 831 ,O mg of potassium allylglucosinolate
monohydrate in water in a 100 ml one-mark
Check the purity in accordance with the procedure
volumetric flask. Make up to the mark with water.
described in 4.5.1.3.
The Solution thus prepared may be stored in a
4.5.2.4 Response factor
refrigerator at approximately 4 “C for up to a week
or in a freezer at - 18 “C for a longer period.
Verify that the response factors of glucotropaeolin,
in comparison with sinigrin, correspond to those in-
dicated in 9.2.
4.5.1.3 Purity check
4.6 Mobile phases
Use one or more of the following three tests:
4.6.1 Eluant A: water, purified by passing it through
-
HPLC analysis using the method specified in this
an activated charcoal cartridge (e.g. Norganic
part of ISO 9167;
Millipore’) System) or water of equivalent purity.
-
analysis of the intact sinigrin by HPLC (ion-pair
HPLC
4.6.2 Eluant B: acetonitrile, grade,
technique);
20 % (1/‘/1/) Solution in purified water. The concen-
tration may be modified in relation to the column
-
analysis of the desulfated and silylated sinigrin
used.
by gas chromatography.
4.7 Ion-exchange resin, use either 4.7.1 or 4.7.2.
For each test, the chromatogram shall show only
one major peak representing at least 98 % of the
total peak area. 4.7.1 DEAE Sepharose CL-6B2) Suspension, avail-
able commercially ready for use, or an equivalent
Confirm the purity by determining the quantity of
product.
glucose released after hydrolysis with myrosinase
(thioglucoside glucohydrolase, EC 3.2.3.1). Measure
4.7.2 DEAE Sephadex A2!j2) Suspension, prepared
the glucose by enzymatic means. The use of a
as follows.
commercially available test kit facilitates the deter-
Mix 10 g of DEAE Sephadex A25 resin (or an equiv-
mination. Take into account any free glucose pres-
alent resin) in excess 2 mol/1 acetic acid solution.
ent; this is determined in the Same way but without
Leave to settle. Add 2 mol/1 acetic acid until the
addition of myrosinase. The molar concentration of
volume of the liquid is equal to twice the volume of
glucose measured should be at least 98 % of the
the Sediment.
molar concentration of the sinigrin Solution tested.
1) Norganic Millipore System is an example of a suitable product available commercially. This information is given for the
convenience of users of this part of ISO 9167 and does not constitute an endorsement by ISO of this product.
products avai lable commercially. This information is given for
2) DEA Sepharo se and S ephadex are exam ples of su itable
of this part of ISO 9167 and not consti tute an endorsement by ISO of these products.
the conv ,enience of users does
2
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SIST EN ISO 9167-1:1998
ISO 91674:1992(E)
4.8 Sulfatase, Helix pomafia type Hl (EC 3.1.6.1), Pour 3 ml of the 5 mmol/l sinigrin Solution (4.5.1.1)
having an activity of greater than 0,5 units of activity into a 100 ml one-mark volumetric flask and make
per millilitre of purified sulfatase solution. up to the mark with Solution c).
Purify, test and dilute the sulfatase in accordance
4.8.3.2 Test of activity
with the method described in 4.8.1 to 4.8.4.
Using a pipette, transfer 2 ml of the buffered sinigrin
solution (4.8.3.1) into the reference and measuring
4.8.1 Preparation of ion-exchange columns
cells of the spectrometer (5.3) adjusted to a wave-
Cut five Pasteur pipettes (5.9) 7 cm above the neck length of 229 nm with a cell temperature of 30 “C.
and place a glass wool plug (5.8) in the neck. Place At time t = 0, add 50 pl of purified sulfatase (4.8.2) to
the pipettes vertically on a stand and add to each a the measuring cell and immediately switch on the
sufficient quantity of ion-exchange resin (4.7) such recorder. Stop the recorder when the absorbance
that, once the water has drained Off, a volume of no longer varies (ii,), plot the tangent to the Point
500 pl of resin is obtained. I = 0 and measure its gradient An/Al.
The activity of the sulfatase (i.e. the production of
Pour 1 ml of the imidazole formate Solution (4.4) into
each pipette and rinse twice with 1 ml portions of 1 micromole of desulfated sinigrin per minute at
water. 30 “C and pH 5,8), expressed in units of activity per
millilitre of sulfatase solution, is equal to
4.8.2 Purification
AA
V 1000 x 106
AZ xxi-x 50
Weigh, to the nearest 0,l mg, 25 mg of Helix poniatia
type Hl (4.8), dissolve it in 2,5 ml of water and
transfer 500 pl of this Solution to each of the columns
AA/AI is the gradient of the tangent to the
prepared in 4.8.1. Wash each column with 1,5 ml of
Point t = 0, in absorbance units per
water and discard the effluent. Then add 1,5 ml of
minute;
the sodium acetate Solution (4.3) and collect the
eluates from the five columns in a test tube.
V is the volume, in litres, of the reacting
medium (i.e. 2,05 x 10we3 1);
Concentrate the eluates by filtration using a
Millipore PTGC 11K253) immersion filter until ap-
AF (approximately 1 500 I.mol- ‘vcm- ‘) is
proximately 100 ~1 of liquid remains (sulfatase with
the differente between the molar ex-
a molar mass in excess of 5 000 is not removed).
tinction coefficients of sinigrin and of
Add 2,5 ml of water and concentrate once more by
desulfosinigrin at 228 nm, i.e.
filtration until approximately 100 pl of liquid remains.
n
Dilute to 2,5 ml with water and store the purified
AF, = .-!f!-
sulfatase in a freezer at - 18 OC in small amounts in *
k /
Order to allow defrosting of the amount necessary
where
for immediate use.
,& is the differente between the
4.8.3 Test of the sulfatase activity
absorbance at equilibrium of
the desulfated sinigrin and the
4.8.3.1 Preparation of a 0,15 mmol/l sinigrin sol- absorbance at time t = 0;
ution, buffered to pH 58.
I is the path length of the cell, in
centimetres (i.e. 1 cm);
Prepare three solutions in succession as follows:
is the concentration of desul-
c
a) transfer- 1 ml of acetic acid to a 500 ml one-mark
fated sinigrin at equilibrium, in
volumetric flask and make up to the mark with
moles per litre, i.e.
water;
0,15 x 1o--3 x 0,95 x 2
b) transfer 1 ml of ethylene diamine to a 500 ml c=-
2,05 -
one-mark volumetric flask and make up to the
mark with water;
= 1,39 x 10 mol/1
c) mix 73 ml of Solution a) with 40 ml of Solution b) 0,95 is the yield at equilibrium
and adjust the mixture to pH 5,8 using Solution of the desulfatation of the
sinigrin.
a) or Solution b) as appropriate.
3) Millipore PTGC 11K25 is an example of a suitable product available commercially. This information is given for the
convenience sf users of this part of ISO 9167 and does not constitute an endorsement by ISO of this product.
3
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